Growth inhibitor for leukemia cells comprising antisense oligonucleotide derivative to wilms tumor gene (wt1)

ABSTRACT

This invention provides a growth inhibitor for leukemia cells comprising an antisense oligonucleotide derivative to Wilms&#39; tumor gene (WT1).

This application is a divisional of application Ser. No. 08/952,664,filed Dec. 1, 1997, now U.S. Pat. No. 6,034,235, which is a NationalStage of PCT/JP96/01394, filed May 24, 1996.

TECHNICAL FIELD

The present invention relates to a growth inhibitor for leukemia cellscomprising an antisense nucleotide derivative.

BACKGROUND ART

Wilms' tumor is a pediatric kidney tumor that occurs as a result ofdeactivation of both allele of the Wilms' tumor gene (WT1) located onchromosome 11p13 (Call, K. M., et al., Cell 60: 509, 1990). A non-codingupstream sequence of WT1 (C. E. Campbell, et al., Oncogene 9: 583-595,1994) and a coding region that includes the intron (D. A. Haber, et al.,Proc. Natl. Acad. Sci. USA, 88: 9618-9622, 1991) have previously beenreported, and they are expected to be involved in the growth anddifferentiation of the tumor and so forth (D. A. Haber, et al., Proc.Natl. Acad. Sci. USA, 88: 9618-9622, 1991).

However, it was not known that WT1 is involved in the growth of leukemiacells, and that an antisense oligonucleotide derivative to WT1suppresses and inhibits growth of leukemia cells.

DISCLOSURE OF THE INVENTION

Thus, the present invention provides a growth inhibitor for leukemiacells comprising an antisense nucleotide derivative to Wilms' tumor gene(WT1).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibitory effects of oligonucleotide onthe growth of leukemia cell line K562.

FIG. 2 is a graph showing the relationship between the concentrations ofoligonucleotides SE3 and AS3 and the growth of leukemia cell line K562.

FIG. 3 is a graph showing the relationship between the concentrations ofoligonucleotides SE4 and AS4 and the growth of leukemia cell line K562.

FIG. 4 is a graph showing the time-based effects of oligonucleotides SE3and AS3 on the growth of leukemia cell line K562.

FIG. 5 is a graph showing the effects of various oligonucleotides on thegrowth of leukemia cell line K562.

FIG. 6 is a graph showing the inhibitory effects of variousoligonucleotides on the growth of leukemia cell line HEL positive forexpression of WT1.

FIG. 7 is a graph showing the inhibitory effects of variousoligonucleotides on the growth of leukemia cell line THP-1 positive forexpression of WT1.

FIG. 8 is a graph showing the inhibitory effects of various nucleotideson the growth of malignant lymphoma cell line U937 negative forexpression of WT1.

FIG. 9 is a graph showing the effects of oligonucleotides SE3 and AS3 onthe formation of leukemia cell colonies from bone marrow mononuclearcells derived from leukemia patients.

FIG. 10 is a graph showing the effects of oligonucleotides SE3 and AS3on the formation of granulocytic macrophage colonies from bone marrowmononuclear cells derived from healthy volunteers.

FIGS. 11A-11B, panel 11A is a photograph of the results ofelectrophoresis indicating a decrease in the level of WT1 protein incells in the case of adding various WT1 antisense oligonucleotides to aculture of K562 cells; panel 11B indicates a decrease in the level ofWT1 protein in cells in the case of adding WT1 antisenseoligonucleotides to fresh leukemia cells from a patient with AML.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a leukemia cell growth inhibitorcomprising an antisense oligonucleotide derivative to WT1. The antisenseoligonucleotide derivatives used in the present invention is anantisense oligonucleotide derivative to WT1, examples of which includethat to the transcription capping site of WT1, gene that to thetranslation starting region, that to an exon or that to an intron.

For example, a nucleotide sequence of a sense DNA strand of the regioncontaining the transcription capping site of WT1 is represented with SEQID NO: 9. In addition, a nucleotide sequence of a sense DNA strand ofexons 1 to 10 of the region coding for WT1 is represented with SEQ IDNOS: 10 to 19. The present invention uses an antisense oligonucleotidederivative to such a nucleotide sequence of the sense DNA strand of WT1.This antisense oligonucleotide derivative is an antisenseoligonucleotide derivative comprising 5 to 50 continuous nucleotides andpreferably 9 to 30 nucleotides of antisense DNA or RNA chain for WT1, or5 to 70 nucleotides and preferably 9 to 50 nucleotides intermittently orpartially complementary to DNA or RNA chain for WT1 and capable ofbinding to DNA or RNA chain for WT1.

Examples of antisense oligonucleotide derivatives to the transcriptioncapping site include those having the following nucleotide sequences:5′-AGGGTCGAATGCGGTGGG-3′ (SEQ ID NO: 2) and 5′-TCAAATAAGAGGGGCCGG-3′(SEQ ID NO: 4). In addition, examples of antisense oligonucleotidederivatives to the translation starting region include antisenseoligonucleotide derivatives to the translation starting codon ATG andits upstream and/or downstream region such as the following nucleotidesequence: 5′-GTCGGAGCCCATTTGCTG-3′ (SEQ ID NO: 6).

In addition, ten exons are contained in the region coding for WT1, andexamples of the antisense oligonucleotide derivative of the presentinvention include those to the sequences contained in any of theseexons, those to the sequences extending over any two consecutive exonsafter splicing or those to the sequences extending over a consecutiveintron and exon, and those to sequences of all introns and the 3′ and 5′non-coding regions. One example of an antisense oligonucleotidederivative is that to the 6th exon, an example of which is that to thefollowing nucleotide sequence: 5′-CGTTGTGTGGTTATCGCT-3′ (SEQ ID NO: 8).

Moreover, although there are no particular restrictions on the regioncorresponding to the antisense oligonucleotide derivative of the presentinvention having a nucleotide sequence intermittently or partiallycomplementary to the DNA or RNA chain for WT1, those similar toribozymes having a function to cleave the DNA chain or RNA chain for WT1are included in these.

The structure of antisense olignucleotide derivative used in the presentinvention is as shown in chemical formula 1, wherein X may independentlybe an oxygen (O), sulfur (O), lower alkyl group, primary amine orsecondary amine. Y may independently be an oxygen (O) or sulfur (S). Zis a hydrogen atom or hydroxyl group. B is chosen from adenine, guanine,thymine or cytosine when Z is a hydrogen atom, or chosen from adenine,guanine, uracil or cytosine when Z is a hydroxyl group, and is mainly anoligonucleotide complementary to DNA or mRNA coding for WT1. R isindependently a hydrogen atom, dimethoxytrityl group or lower alkylgroup. N is an integer of 7-28.

Preferable examples of antisense oligonucleotide derivatives include notonly non-modified antisense oligonucleotides, but also modifiedantisense oligonucleotides. Examples of these modified forms include lowalkyl phosphonate forms like the above-mentioned methylphosphonate formor ethylphosphonate form, and other phosphorothioate forms orphosphoroamidate forms (see chemical formula 2).

These antisense oligonucleotide derivatives can be obtained according tothe following conventional methods.

The antisense oligonucleotides in which X and Y in chemical formula 1are O and Z is a hydrogen atom or hydroxyl group are easily synthesizedby a commercially available DNA synthesizer (for example, thatmanufactured by Applied Biosystems).

Antisense oligodeoxyribonucleotide in which Z is a hydrogen atom can beobtained by a method such as solid phase synthesis usingphosphoroamidite or solid phase synthesis using hydrogen phosphonate.

See, for example, T. Atkinson and M. Smith in Oligonucleotide Synthesis:A Practical Approach, ed. M. J. Gait, IRL Press, 35-81 (1984); M. H.Caruthers, Science, 230, 281 (1985); A. Kume, M. Fujii, M. Sekine and M.Hata, J. Org. Chem., 49, 2139 (1984); B. C. Froehler and M. Matteucci,Tetrahedron Lett., 27, 469 (1986); P. J. Garegg, I. Lindh, T. Regberg,J. Stawinski, R. Stromberg and C. Henrichson, ibid., 27, 4051 (1986); B.S. Sproat and M. J. Gait in Oligonucleotide Synthesis: A PracticalApproach, ed. M. J. Gait, IRL Press, 83-115 (1984); S. L. Beaucage andM. H. Caruthers, Tetrahedron Lett., 22, 1859-1862 (1981); M. D.Matteucci and M. H. Caruthers, Tetrahedron Lett., 21, 719-722 (1980);and, M. D. Matteucci and M. H. Caruthers, J. Am. Chem. Soc., 103,3185-3191 (1981).

Triester phosphate modified forms, in which X is a lower alkoxy group,can be obtained by ordinary methods, such as treatment of anoligonucleotide obtained by chemical synthesis with a tosylchloridesolution of DMF, methanol and 2,6-lutidine (Moody H. M., et al., NucleicAcids Res., 17, 4769-4782 (1989).

Alkylphosphonate modified forms, in which X is an alkyl group, can beobtained by ordinary methods using, for example, phosphoamidite (M. A.Dorman, et al., Tetrahedron, 40, 95-102 (1984); and, K. L. Agarwal andF. Riftina, Nucleic Acids Res., 6, 3009-3024 (1979)).

Phosphorothioate modified forms in which X is S can be obtained byordinary methods such as solid phase synthesis using sulfur (C. A.Stein, et al., Nucleic Acids Res., 16, 3209-3221 (1988) or solid phasesynthesis using tetraethylthiolam disulfide (H. Vu and B. L. Hirschbein,Tetrahedron Letters, 32, 3005-3008 (1991)).

Phosphorodithioate modified forms in which X and Y are both S can beobtained by, for example, solid phase synthesis by convertingbis-amidite to thioamidite and allowing sulfur to act on the thioamidite(W. K. D. Brill, et al., J. Am. Chem. Soc., 111, 2321-2322 (1989)).

Phosphoroamidate modified forms in which X is a primary amine orsecondary amine can be obtained by, for example, solid phase synthesisby treating hydrogen phosphonate with a primary or secondary amine (B.Froehler, et al., Nucleic Acids Res., 16, 4831-4839 (1988)), or byoxidizing amidite with tert-butyl hydroperoxide (H. Ozaki, et al.,Tetrahedron Lett., 30, 5899-5902 (1989)).

Although synthesis of antisense oligoribonucleotide in which Z is ahydroxyl group is extremely difficult in comparison with synthesis ofantisense oligodeoxyribonucleotide since the 2′-hydroxyl group on ribose(sugar) must be protected, it can be synthesized by suitably selectingthe protecting group and phosphorylation method (see, Basic MicrobiologyCourse, Vol. 8, Genetic Engineering, E. Ohtsuka, K. Miura, ed. T. Andoand K. Sakaguchi, Oct. 10, 1987, Kyoritsu Publishing Co., Ltd.).

Purification and confirmation of purity can be performed byhigh-performance liquid chromatography and polyacrylamide gelelectrophoresis. Confirmation of molecular weight can be performed byElectrospray Ionization Mass Spectrometry or Fast Atom Bombardment-MassSpectrometry.

The antisense oligonucleotide derivatives of the present invention actsat any stage from genomic DNA to mature mRNA, and suppression of itsexpression is thought to inhibit growth of leukemia cells. Thus, theantisense oligonucleotides of the invention of the present applicationis expected to be effective in the treatment of leukemia.

Moreover, as will be described later, the antisense oligonucleotidederivatives of the present invention is thought to specifically inhibitleukemia cells without inhibiting the growth of normal bone marrowcells. Thus, it can also be applied to “autologous bone marrowtransplantation” and “autologous peripheral blood stem celltransplantation” in which, for example, after first removing bone marrowcells or peripheral blood stem cells from the body and treating them invitro with the antisense oligonucleotide derivatives of the presentinvention to inhibit the growth of leukemia cells, only normal bonemarrow cells or normal peripheral blood stem cells are returned to thebody.

The antisense oligonucleotide derivatives of the present invention canalso be used in the form of an external preparation such as a linimentor poultice by mixing with a suitable inactive base.

In addition, the antisense oligonucleotide derivatives of the presentinvention can also be used in the form of tablets, powders, granules,capsules, liposome capsules, injection preparations, liquids or nosedrops by adding a vehicle, isotonic agent, solubility assistant,stabilizer, preservative or analgesic and so forth as necessary, or canbe made into a freeze-dried preparation. These formulations can beprepared in accordance with routine methods.

The antisense oligonucleotide derivatives of the present invention canbe applied directly to the affected area of the patients, or applied soas to be able to reach the affected area as a result of intravascularadministration and so forth. Moreover, antisense inclusion materials canalso be used to improve duration and membrane permeation. Examples ofthese include liposomes, poly-L-lysine, lipids, cholesterol lipofectinand their derivatives.

The dose of the antisense oligonucleotide derivative of the presentinvention is such that a preferable amount can be used by suitablypreparing a dose according to the patient's condition, age, sex and bodyweight. In addition, the administration method can be suitably selectedfrom various administration methods, including oral administration,intramuscular administration, intraperitoneal administration,intradermal administration, subcutaneous administration, intravenousadministration, intraarterial administration and rectal administrationaccording to the patient's conditions, the drug forms and so forth.

The following provides a detailed explanation of the present inventionthrough Examples.

EXAMPLES Synthesis Example 1

The oligodeoxyribonucleotides used below (SEQ ID NOS: 1 to 8) weresynthesized using an automatic synthesizer (Applied Biosystems),purified by high-performance liquid chromatography, precipitated threetimes with ethanol, and suspended in phosphate buffer. The synthesizedoligonucleotides were as listed below.

SEQ ID NO: 1: Sense sequence of transcription capping site (SE1)

SEQ ID NO: 2: Antisense sequence of transcription capping site (AS1)

SEQ ID NO: 3: Sense sequence of transcription capping region (SE2)

SEQ ID NO: 4: Antisense sequence of transcription capping region (AS2)

SEQ ID NO: 5: Sense sequence of translation starting region (SE3)

SEQ ID NO: 6: Antisense sequence of translation starting region (AS3)

SEQ ID NO: 7: Sense sequence of exon 6 (SE4)

SEQ ID NO: 8: Antisense sequence of exon 6 (AS4)

Example 1

5×10⁴ cells/ml of leukemia cell line K562 positive for WT1 expressionwere inoculated into RPMI 1640 medium not containing fetal calf serum(FCS) contained in the wells of a flat-bottom 96-well plate in theamount of 100 μl/well. Each oligonucleotide was added to a series ofthree wells to a final concentration of 200 μg/well. After incubatingfor 2 hours, FCS was added to each well to a final concentration of 10%.Oligonucleotides were then added to the culture in an amount equal tohalf the above-mentioned amount every 24 hours.

After culturing for 96 hours, the numbers of viable cells were countedusing the pigment elimination method. An equal volume of PBS notcontaining nucleotide was added as the control culture, and the numberof cells of this control culture was taken to be 100%.

The results are shown in FIG. 1. As is clear from this figure, all ofthe antisense oligonucleotides powerfully inhibited cell growth incomparison with the corresponding sense oligonucleotides.

Example 2

The same experiment as that described in Example 1 was performed, butoligonucleotides SE3 and AS3 were added at various concentrations. As isclear from FIG. 2, although sense oligonucleotide (SE3) virtually didnot inhibit cell growth, antisense oligonucleotide (AS3) inhibited cellgrowth in a dose dependent manner.

Example 3

The same experiment as that described in Example 1 was performed, butoligonucleotides SE4 and AS4 were added at various concentrations. As isclear from FIG. 3, although sense oligonucleotide (SE4) virtually didnot inhibit cell growth, antisense oligonucleotide (AS4) inhibited cellgrowth in a dose dependent manner.

Example 4

The same experiment as described in Example 1 was performed. However,the cells were cultured in a flat-bottom 24-well plate at aconcentration of 5×10⁴ cells/ml and in the amount of 1 ml/well.Oligonucleotides SE3 and AS3 were added and the numbers of viable cellswere counted daily for 2 to 5 days. The results are shown in FIG. 4. Asis clear from the figure, although cell growth similar to the controlwas observed in the case of adding sense oligonucleotide, in the case ofadding antisense oligonucleotide, cell growth was inhibited.

Example 5

The same experiment as described in Example 1 was performed. However,SE3, AS3, an antisense oligonucleotide 5′-AGAGAAGAAGGGAACCCC-3′ (SEQ IDNO: 20) (MPO-AS) to myeloperoxidase (MPO) gene, and an antisenseoligonucleotide 5′-GCGTGGGCAGCCTGGGAA-3′ (SEQ ID NO: 21) (FV-AS) toblood coagulation factor V (FV) were used for the oligonucleotides. Asis clear from FIG. 5, cell growth was inhibited only in the case ofusing AS3.

Example 6

The same experiment as described in Example 1 was performed, but WT1expression-positive cell lines HEL and THP-1 as well as WT1expression-negative cell line U937 were used as the experimental cells.The same eight types of oligonucleotides used in Example 1 were used asthe oligonucleotides. In the case of using WT1 expression-positive cellline HEL (FIG. 6) or THP-1 (FIG. 7), cell growth was inhibited byantisense oligonucleotide. In contrast, in the case of using WT1expression-negative cell line U937 (FIG. 8), cell growth was notinhibited even if antisense oligonucleotide was added.

Example 7

Bone marrow cells from leukemia patients and healthy volunteers weretreated with heparin and suspended in RPMI 1640 medium to obtain bonemarrow mononuclear cells by Ficoll-Hypaque density gradientcentrifugation. A protein (100 μl/well) of the above-mentionedmononuclear cells at a cell density of 1.5×10⁶ cells/ml were added to aflat-bottom 96-well plate containing A-MEM containing GM-CSF (100 ng/ml)and IL-3 (100 units/ml). Treatment with oligonucleotides (SE3 and AS3)was performed in the same manner as Example 1.

After 96 hours, the cells were collected and plated in methylcellulosemedium [1.2% methylcellulose α-MEM, 20% FCS, GM-CSF (100 ng/ml), G-CSF(100 ng/ml), IL-3 (100 units/ml) and SCF (10 ng/ml)]. Culturing wasperformed in three series. The number of leukemia cell colonies (CFU-L)and granulocytic macrophage colonies (CFU-GM) were counted on day 14.

FIG. 9 shows the morphology of the leukemia colonies in samples fromfour leukemia patients (two acute myeloid leukemia (AML) patients and 2chronic myeloid leukemia (CML) patients). The formation of colonies canbe seen to be inhibited by antisense oligonucleotide. FIG. 10 shows theappearance of granulocytic macrophage colonies in samples from healthyvolunteers. Colony formation is not inhibited by either of the antisenseoligonucleotides.

Example 8

Random oligonucleotide, oligonucleotide AS1, oligonucleotide AS2 oroligonucleotide AS3 was added at a concentration of 200 μg/ml to K562cells (A) or fresh leukemia cells from a patient with AML (B) at a celldensity of 5×10⁴ cells/well in a 24-well plate, followed by addition ofthe oligonucleotides at a concentration of 100 μg/ml every 24 hours. Thecells were harvested 4 days after the initial treatment witholigonucleotide, washed with PBS and lysed with Laemli sample buffer.

Each cell lysate from 2×10⁴ cells was boiled for 5 minutes, and thenapplied to each lane of 5% dodecylsodium sulfate-polyacrylamide gel.Following electrophoresis, the proteins were transferred to an Immobilonpolyvinylidene difluoride filter (Millipore Corp. Massachusetts, USA).This filter was then probed using an anti-WT1 polyclonal antibody tosynthetic polypeptide (amino acid positions 177 to 192: Lys His Glu AspPro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln (SEQ ID NO: 22)). Thiswas followed by treatment with horseradish peroxidase-boundanti-immunoglobulin antibody (Amersham, Little Chalfont, U.K.). Afterwashing, the filter was immersed in detection reagent (Amersham, LittleChalfont, U.K.) for 1 hour followed by autoradiography treatment for 1to 5 minutes.

After washing twice with TBST (Tris buffer containing 0.05% Tween 20),the filter was probed with anti-actin monoclonal antibody (OncogeneScience Inc., New York, USA) followed by autoradiography in the mannerdescribed above.

The density of the bands corresponding to WT1 protein and actin weremeasured with a CS-9000 densitometer (Shimizu, Kyoto) followed bycalculation of the WT1/actin ratio.

The results are shown in FIGS. 11A and B. In these figures, lane 1 showsthe results in the case of adding random oligonucleotide, lane 2 thecase of adding oligonucleotide AS3, lane 3 the case of addingoligonucleotide AS1, and lane 4 the case of adding oligonucleotide AS2.In these figures, A indicates the results in the case of using K562cells, while B indicates those in the case of using fresh leukemia cellsfrom a patient with AML.

As is clear from FIG. 11A, in the case of adding WT1 oligonucleotide tomedium containing K562 cells, the level of WT1 protein decreasedsignificantly. On the other hand, control in the form of randomnucleotide did not affect the level of WT1 protein. In addition, as isclear from FIG. 11B, in the case of adding WT1 oligonucleotide to mediumcontaining leukemia cells recently isolated from a patient with AML, thelevel of WT1 protein decreased significantly. These results clearlyshowed that WT1 antisense oligonucleotide specifically inhibits thegrowth of leukemia cells by decreasing the level of WT1 protein.

Industrial Applicability

As has been stated above, the antisense oligonucleotides of the presentinvention is effective in inhibiting the growth of leukemia cells, andis therefore expected to be useful as a novel leukemia treatment.

22 18 base pairs nucleic acid single linear other nucleic acid /desc =“Synthetic DNA” not provided 1 CCCACCGCAT TCGACCCT 18 18 base pairsnucleic acid single linear other nucleic acid /desc = “Synthetic DNA”not provided 2 AGGGTCGAAT GCGGTGGG 18 18 base pairs nucleic acid singlelinear other nucleic acid /desc = “Synthetic DNA” not provided 3CCGGCCCCTC TTATTTGA 18 18 base pairs nucleic acid single linear othernucleic acid /desc = “Synthetic DNA” not provided 4 TCAAATAAGA GGGGCCGG18 18 base pairs nucleic acid single linear other nucleic acid /desc =“Synthetic DNA” not provided 5 CAGCAAATGG GCTCCGAC 18 18 base pairsnucleic acid single linear other nucleic acid /desc = “Synthetic DNA”not provided 6 GTCGGAGCCC ATTTGCTG 18 18 base pairs nucleic acid singlelinear other nucleic acid /desc = “Synthetic DNA” not provided 7AGCGATAACC ACACAACG 18 18 base pairs nucleic acid single linear othernucleic acid /desc = “Synthetic DNA” not provided 8 CGTTGTGTGG TTATCGCT18 1272 base pairs nucleic acid single linear other nucleic acid /desc =“Synthetic DNA” not provided 9 TGGTATCCTC GACCAGGGCC ACAGGCAGTGCTCGGCGGAG TGGCTCCAGG AGTTACCCGC 60 TCCCTGCCGG GCTTCGTATC CAAACCCTCCCCTTCACCCC TCCTCCCCAA ACTGGGCGCC 120 AGGATGCTCC GGCCGGAATA TACGCAGGCTTTGGGCGTTT GCCAAGGGTT TTCTTCCCTC 180 CTAAACTAGC CGCTGTTTTC CCGGCTTAACCGTAGAAGAA TTAGATATTC CTCACTGGAA 240 AGGGAAACTA AGTGCTGCTG ACTCCAATTTTAGGTAGGCG GCAACCGCCT TCCGCCTGGC 300 GCAAACCTCA CCAAGTAAAC AACTACTAGCCGATCGAAAT ACGCCCGGCT TATAACTGGT 360 GCAACTCCCG GCCACCCAAC TGAGGGACGTTCGCTTTCAG TCCCGACCTC TGGAACCCAC 420 AAAGGGCCAC CTCTTTCCCC AGTGACCCCAAGATCATGGC CACTCCCCTA CCCGACAGTT 480 CTAGAGCAAG AGCCAGACTC AAGGGTGCAAAGCAAGGGTA TACGCTTCTT TGAAGCTTGA 540 CTGAGTTCTT TCTGCGCTTT CCTGAAGTTCCCGCCCTCTT GGAGCCTACC TGCCCCTCCC 600 TCCAAACCAC TCTTTTAGAT TAACAACCCCATCTCTACTC CCACCGCATT CGACCCTGCC 660 CGGACTCACT GCTACTGAAC GGACTCTCCAGTGAGACGAG GCTCCCACAC TGGCGAAGGC 720 AAGAAGGGGA GGTGGGGGGA GGGTTGTGCCACACCGGCCA GCTGAGAGCG CGTGTTGGGT 780 TGAAGAGGAG GGTGTCTCCG AGAGGGACGCTCCCTCGGAC CCGCCCTCAC CCCAGCTGCG 840 AGGGCGCCCC CAAGGAGCAG CGCGCGCTGCCTGGCCGGGC TTGGGCTGCT GAGTGAATGG 900 AGCGGCCGAG CCTCCTGGCT CCTCCTCTTCCCCGCGCCGC CGGCCCCTCT TATTTGAGCT 960 TTGGGAAGCT GAGGGCAGCC AGGCAGCTGGGGTAAGGAGT TCAAGGCAGC GCCCACACCC 1020 GGGGGCTCTC CGCAACCCGA CCGCCTGTCGCTCCCCCACT TCCCGCCCTC CCTCCCACCT 1080 ACTCATTCAC CCACCCACCC ACCCAGAGCCGGGACGGCAG CCCAGGCGCC CGGGCCCCGC 1140 CGTCTCCTCG CCGCGATCCT GGACTTCCTCTTGCTGCAGG ACCCGGCTTC CACGTGTGTC 1200 CCGGAGCCGG CGTCTCAGCA CACGCTCCGCTCCGGGCCTG GGTGCCTACA GCAGCCAGAG 1260 CAGCAGGGAG TC 1272 457 base pairsnucleic acid single linear other nucleic acid /desc = “Synthetic DNA;portion of exon 1 of WT1 gene” not provided 10 TCTGAGCCTC AGCAAATGGGCTCCGACGTG CGGGACCTGA ACGCGCTGCT GCCCGCCGTC 60 CCCTCCCTGG GTGGCGGCGGCGGCTGTGCC CTGCCTGTGA GCGGCGCGGC GCAGTGGGCG 120 CCGGTGCTGG ACTTTGCGCCCCCGGGCGCT TCGGCTTACG GGTCGTTGGG CGGCCCCGCG 180 CCGCCACCGG CTCCGCCGCCACCCCCGCCG CCGCCGCCTC ACTCCTTCAT CAAACAGGAG 240 CCGAGCTGGG GCGGCGCGGAGCCGCACGAG GAGCAGTGCC TGAGCGCCTT CACTGTCCAC 300 TTTTCCGGCC AGTTCACTGGCACAGCCGGA GCCTGTCGCT ACGGGCCCTT CGGTCCTCCT 360 CCGCCCAGCC AGGCGTCATCCGGCCAGGCC AGGATGTTTC CTAACGCGCC CTACCTGCCC 420 AGCTGCCTCG AGAGCCAGCCCGCTATTCGC AATCAGG 457 123 base pairs nucleic acid single linear othernucleic acid /desc = “Synthetic DNA; exon 2 of WT1 gene” not provided 11GTTACAGCAC GGTCACCTTC GACGGGACGC CCAGCTACGG TCACACGCCC TCGCACCATG 60CGGCGCAGTT CCCCAACCAC TCATTCAAGC ATGAGGATCC CATGGGCCAG CAGGGCTCGC 120TGG 123 103 base pairs nucleic acid single linear other nucleic acid/desc = “Sythetic DNA; exon 3 of WT1 gene” not provided 12 GTGAGCAGCAGTACTCGGTG CCGCCCCCGG TCTATGGCTG CCACACCCCC ACCGACAGCT 60 GCACCGGCAGCCAGGCTTTG CTGCTGAGGA CGCCCTACAG CAG 103 78 base pairs nucleic acidsingle linear other nucleic acid /desc = “Synthetic DNA; exon 4 of WT1gene” not provided 13 TGACAATTTA TACCAAATGA CATCCCAGCT TGAATGCATGACCTGGAATC AGATGAACTT 60 AGGAGCCACC TTAAAGGG 78 51 base pairs nucleicacid single linear other nucleic acid /desc = “Synthetic DNA; exon 5 ofWT1 gene” not provided 14 AGTTGCTGCT GGGAGCTCCA GCTCAGTGAA ATGGACAGAAGGGCAGAGCA A 51 97 base pairs nucleic acid single linear other nucleicacid /desc = “Synthetic DNA; exon 6 of WT1 gene” not provided 15CCACAGCACA GGGTACGAGA GCGATAACCA CACAACGCCC ATCCTCTGCG GAGCCCAATA 60CAGAATACAC ACGCACGGTG TCTTCAGAGG CATTCAG 97 151 base pairs nucleic acidsingle linear other nucleic acid /desc = “Synthetic DNA; exon 7 of WT1gene” not provided 16 GATGTGCGAC GTGTGCCTGG AGTAGCCCCG ACTCTTGTACGGTCGGCATC TGAGACCAGT 60 GAGAAACGCC CCTTCATGTG TGCTTACCCA GGCTGCAATAAGAGATATTT TAAGCTGTCC 120 CACTTACAGA TGCACAGCAG GAAGCACACT G 151 90 basepairs nucleic acid single linear other nucleic acid /desc = “SyntheticDNA; exon 8 of WT1 gene” not provided 17 GTGAGAAACC ATACCAGTGTGACTTCAAGG ACTGTGAACG AAGGTTTTCT CGTTCAGACC 60 AGCTCAAAAG ACACCAAAGGAGACATACAG 90 93 base pairs nucleic acid single linear other nucleicacid /desc = “Synthetic DNA; exon 9 of WT1 gene” not provided 18GTGTGAAACC ATTCCAGTGT AAAACTTGTC AGCGAAAGTT CTCCCGGTCC GACCACCTGA 60AGACCCACAC CAGGACTCAT ACAGGTAAAA CAA 93 158 base pairs nucleic acidsingle linear other nucleic acid /desc = “Synthetic DNA; portion of exon10 of WT1 gene” not provided 19 GTGAAAAGCC CTTCAGCTGT CGGTGGCCAAGTTGTCAGAA AAAGTTTGCC CGGTCAGATG 60 AATTAGTCCG CCATCACAAC ATGCATCAGAGAAACATGAC CAAACTCCAG CTGGCGCTTT 120 GAGGCGTCTC CCTCGGGGAC CGTTCAGTGTCCCAGGCA 158 18 base pairs nucleic acid single linear other nucleic acid/desc = “Synthetic DNA” not provided 20 AGAGAAGAAG GGAACCCC 18 18 basepairs nucleic acid single linear other nucleic acid /desc = “SyntheticDNA” not provided 21 GCGTGGGCAG CCTGGGAA 18 16 amino acids amino acidlinear peptide not provided 22 Lys His Glu Asp Pro Met Gly Gln Gln GlySer Leu Gly Glu Gln Gln 1 5 10 15

What is claimed is:
 1. A method for treating leukemia comprising, administering ex vivo an antisense oligonucleotide against Wilms' tumor gene, wherein the antisense oligonucleotide is 9-30 nucleotides in length and comprises all or a part of SEQ ID NO: 2, 4, 6 or
 8. 2. A method as set forth in claim 1, wherein said antisense oligonucleotide is an antisense oligonucleotide to at least nine continuing nucleotides at the transcription capping site of the Wilms' tumor gene.
 3. A method as set forth in claim 1, wherein said antisense oligonucleotide is an antisense oligonucleotide to at least nine continuing nucleotides including the translation starting region of the Wilms' tumor gene.
 4. A method as set forth in claim 1, wherein said antisense oligonucleotide is an antisense oligonucleotide corresponding to at least nine continuing nucleotides in an exon of the Wilms' tumor gene. 